Evaluation of the Association of Recombinant Proteins NanH and PknG from Corynebacterium pseudotuberculosis Using Different Adjuvants as a Recombinant Vaccine in Mice.

Caseous lymphadenitis is a chronic contagious disease that causes economic losses worldwide. Treatments are ineffective, thus demonstrating the importance of vaccination. In this study, rNanH and rPknG proteins from Corynebacterium pseudotuberculosis were associated with saponin or aluminum hydroxide adjuvants. Three experimental groups (10 animals each) were immunized with sterile 0.9% saline solution (G1), rNanH + rPknG + Saponin (G2), rNanH + rPknG + Al(OH)3 (G3). The mice received two vaccine doses 21 days apart. Animals were challenged 21 days after the last immunization and evaluated for 50 days, with endpoint criteria applied when needed. The total IgG production levels of the experimental groups increased significantly on day 42 when compared to the control (p < 0.05). When tested against rNanH, G2 had a better rate of anti-rNanH antibodies compared to G3. In the anti-rPknG ELISA, the levels of total IgG, IgG1, and IgG2a antibodies were higher in G2. The vaccines generated partial protection, with 40% of the animals surviving the challenge. The association of recombinant NanH and PknG proteins led to promising protection rates in mice, and although using different adjuvants did not interfere with the survival rate, it influenced the immune response generated by the vaccine formulations.

Immunopotential of Pasteurella multocida bivalent outer membrane protein-based vaccine entrapped in aluminum hydroxide nanoparticles.

Swine pasteurellosis is one of the most economically important diseases of pig caused by Pasteurella multocida (P. multocida) capsular types A and D. These organisms are commensals and opportunistic pathogens in the upper respiratory tract in pig. In the present study, we extracted whole outer membrane proteins (OMP) from P. multocida capsular types A and D and were mixed together in the ratio of 1:1 forming bivalent outer-membrane proteins. The bivalent OMP was adsorbed onto aluminum hydroxide nanoparticles. The size of aluminum hydroxide nanoparticles adsorbed outer membrane protein was found to be in the range of 125 to 130 nm. We observed that aluminum hydroxide nanoparticles adjuvanted bivalent OMP-based vaccine elicited quicker immune kinetics in terms of IgG response as compared to aluminum hydroxide microparticles adjuvanted bivalent bacterin vaccine against P. multocida capsular type A and D.

Testing Novel Inactivation Methods and Adjuvants for Vaccines Against Streptococcus agalactiae in Nile Tilapia Oreochromis niloticus.

Inactivation by hydrogen peroxide and pH manipulation are two novel methods used recently in experimental vaccines against Streptococcus agalactiae in Nile tilapia. Here we describe in detail inactivation using novel methods as well as the classical method of inactivation. These vaccines showed similar moderate efficacy when compared to the conventional formaldehyde vaccine. In addition, we describe the inclusion of adjuvants in a hydrogen peroxide vaccine.

Evaluation of genotoxic and cytogenetic effects of saponins and aluminum hydroxide adjuvant on viral vaccine

Bovine papillomavirus (BPV) is the etiological agent of bovine papillomatosis, infectious disease characterized by the presence of benign tumors that can progress to malignancy. The phylogenetic classification of the PVs is performed based on the sequence homology of the Open Reading Frame L1, the most conserved among different viral serotypes. Given the immunogenicity of saponins, it,s has been used as a candidate as adjuvant use. For this reason, the safety of using saponin as an adjuvant has to be better determined to human or veterinary vaccine use. So, this study aimed to evaluate the mutagenic and genotoxic effect of saponins in comparison with the adjuvant widely used aluminum hydroxide using an isolated and purified L1 protein from BPV as model. In this study, genomic lesions, which after processed without repair can result in mutations, were detected by comet assay. Possible damages to genetic material caused by structural chromosomal changes (clastogenesis), as well as chromosomal losses (aneugenesis) were evaluated by the micronucleus test. Both tests were done on polychromatic erythrocytes and Vero cells. The evaluation of apoptosis and necrosis of treated Vero cells was made by Annexin V / PI staining and flow cytometry. The two vaccine products (L1 + Saponin and L1 + Aluminum Hydroxide) showed damages compatible with the positive control in the comet assay and both slightly elevated the micronucleus levels, in the Cell Viability Assay the results with Aluminum Hydroxide were satisfactory, characterizing Aluminum Hydroxide as a safer adjuvant according to the proposed tests, better than the saponins. Some fractions of the saponin extract separated by High Performance Liquid Chromatography were evaluated against genotoxic activity by comet assay, and their identities were confirmed by similarity to the reference standard by mass spectrometry.

Efficacy of two adjuvants administrated with a novel hydrogen peroxide-inactivated vaccine against Streptococcus agalactiae in Nile tilapia fingerlings.

Streptococcus agalactiae is considered the main bacterial pathogen in cultured Nile tilapia. Formaldehyde-inactivated vaccines are the most accepted method for prevention and control of the disease. However, alternative inactivation methods for S. agalactiae vaccines have not been fully explored. Recently, we developed a hydrogen peroxide-inactivated vaccine against S. agalactiae with moderate efficacy, with the possibility to improve vaccine efficacy by adding adjuvants. The current study compared the efficacy of aluminum hydroxide and Freund's incomplete adjuvant (FIA) incorporated into a novel hydrogen peroxide-inactivated intraperitoneal vaccine against S. agalactiae for Nile tilapia fingerlings. The relative percentage survival (RPS) for aluminum hydroxide-adjuvanted vaccine (59.3%), and FIA-adjuvanted vaccine (77.8%) were higher than the vaccine without adjuvant (40.7%). In addition, fish immunized with aluminum hydroxide-adjuvanted vaccine had significantly higher levels of specific antibodies than control fish at 4 weeks post vaccination (wpv). Blood lymphocytes counts showed a decrease in vaccinated groups when compared to control fish, suggesting white cells migration to the tissues where antigen presentation is ongoing. Fish that received FIA-adjuvanted vaccine exhibited persistence of adjuvant deposits on intraperitoneal surfaces for at least 4 wpv that may be related to its superior performance compared to aluminum hydroxide adjuvanted vaccine, which did not evidence any type of deposit at any sampling times. The results observed in this study demonstrate that hydrogen peroxide-inactivated vaccine administered with either aluminum hydroxide or FIA induce optimal levels of protection, with a superior performance for FIA vaccine, which could be a good alternative to conventional formaldehyde-inactivated vaccines against S. agalactiae, due to its shorter manufacture time, and less toxicity.

Albumin-biomineralized nanoparticles to synergize phototherapy and immunotherapy against melanoma.

To date, cancer phototherapy remains as an unsatisfactory method of cancer treatment due to the high probability of cancer recurrence - an effect that is partly driven by tumor-driven immunosuppression. Therefore, we propose inducing adequate immune responses after photo tumor ablation may be critical to achieve a long term therapeutic effect of phototherapy. Here, we engineered the photosensitizer chlorin e6 (Ce6) and the time-honored immunoadjuvant aluminum hydroxide into bovine serum albumin by albumin-based biomineralization as a novel nanosystem (Al-BSA-Ce6 NPs). After intravenous injection, the nanoparticles not only destroyed tumor cells effectively but also protected animals against tumor rechallenge and metastasis by strongly inducing a systemic anti-tumor immune response. Subsequent analysis demonstrated T cells accumulated in lymph nodes and infiltrated the tumor site, elevating levels of immune indicators including serum antibody, cytokine level and higher proportions of cytotoxic T cells and Th1 cells. These protective effects were not observed with commercially available alumina gels, or when the aluminum hydroxide in the nanoparticles was replaced with ferric hydroxide. Therefore, we present Al-BSA-Ce6 NPs as a novel and unique system for alumina adjuvants that serves as an effective approach for cancer therapy.

Aluminum hydroxide nebulization-induced redox imbalance and acute lung inflammation in mice.

Purpose: Aluminum is the third most abundant metal in the earth's crust and is widely used in industry. Chronic contact with aluminum results in a reduction in the activity of electron transport chain complexes, leading to excessive production of reactive oxygen species (ROS) and oxidative stress. This study aimed to evaluate the effects of short-term exposure of aluminum hydroxide on oxidative stress and pulmonary inflammatory response.Materials and methods: Male BALB/c mice were divided into three groups: control group (CG); phosphate buffered saline group (PBSG) and aluminum hydroxide group (AHG). CG was exposed to ambient air, while PBSG and AHG were exposed to PBS or aluminum hydroxide solutions via nebulization, three times per day for five consecutive days. Twenty-four hours after the last exposure, all animals were euthanized for subsequent analysis.Results: Exposure to aluminum hydroxide in the blood resulted in lower platelet levels, higher neutrophils, and lower monocytes compared to CG and PBSG. Aluminum hydroxide promoted the recruitment of inflammatory cells to the lung. Macrophage, neutrophil and lymphocyte counts were higher in AHG compared to CG and PBSG. Protein oxidation and superoxide dismutase activity were higher, while catalase activity and reduced and oxidizes glutathione ratio in AHG were lower compared to CG and PBSG. Furthermore, there was an increase in the inflammatory markers CCL2 and IFN-γ in AHG compared to CG and PBSG.Conclusion: In conclusion, short-term nebulization with aluminum hydroxide induces the influx of inflammatory cells and oxidative stress in adult BALB/c mice.

Assessment of the acid phosphatase CP01850 from Corynebacterium pseudotuberculosis in DNA and subunit vaccine formulations against caseous lymphadenitis / Avaliação da fosfatase ácida recombinante CP01850 de Corynebacterium pseudotuberculosis em formulações vacinais de subunidade e de DNA contra a linfadenite caseosa

The target cp1002_RS01850 from Corynebacterium pseudotuberculosis was used to construct a DNA and recombinant subunit vaccine against caseous lymphadenitis. Recombinant protein rCP01850 was expressed in Escherichia coli using pAE vector, and DNA vaccine was engineered with pTARGET vector. BALB/c mice were divided in five groups containing eight animals each, inoculated with: pTARGET/cp01850 as DNA vaccine (G1); rCP01850 plus Al (OH)3 as recombinant subunit vaccine (G2); pTARGET/cp01850 and a boost with rCP01850 plus Al (OH)3 (G3); pTARGET (G4); or Al (OH)3 (G5). Mice were inoculated and blood samples were collected on days 0, 21, and 42 for the analysis of total IgG, IgG1 and IgG2a by ELISA. In each group, five animals were challenged with Mic-6 C. pseudotuberculosis strain, and three were used for cytokine quantification by qPCR. Although no group has been protected by vaccines against lethal challenge, G2 showed an increase in the survival rate after challenge. Significantly higher levels of IL-4, IL-12, IFN-γ, total IgG, IgG1 and IgG2a were also detected for G2, evidencing a mixed Th1/Th2 immunological profile. In conclusion, despite no protection level provided by different vaccinal strategies using cp1002_RS01850 from C. pseudotuberculosis, G2 developed a Th1/Th2 immune response with an increase in survival rate.(AU)

O alvo cp1002_RS01850 de Corynebacterium pseudotuberculosis foi utilizado para construir uma vacina recombinante de subunidade e de DNA contra a linfadenite caseosa. A proteína recombinante rCP01850 foi expressa em Escherichia coli usando o vetor pAE, e a vacina de DNA foi construída com o vetor pTARGET. Camundongos BALB/c foram divididos em grupos de oito animais, inoculados com: pTARGET/cp01850 como vacina de DNA (G1); rCP01850 e Al (OH)3 como vacina recombinante de subunidade (G2); pTARGET/cp01850 e um boost com rCP01850 e Al (OH)3 (G3); pTARGET (G4); ou Al (OH)3 (G5). Os animais foram inoculados e amostras de sangue foram coletadas nos dias 0, 21, e 42 do experimento para a análise de IgG total, IgG1 e IgG2a por ELISA. De cada grupo, cinco animais foram desafiados com a cepa Mic-6 de C. pseudotuberculosis, e três foram usados para a quantificação de citocinas por qPCR. Apesar de nenhum grupo ter sido protegido pelas vacinas testadas contra o desafio letal, G2 apresentou taxa de sobrevida e níveis de IL-4, IL-12, IFN-γ, IgG total, IgG1 e IgG2a significativamente mais altos, evidenciando um perfil imunológico misto Th1/Th2. Conclui-se que apesar das diferentes estratégias vacinais utilizando cp1002_RS01850 de C. pseudotuberculosis não terem sido capazes de gerar proteção, G2 desenvolveu uma resposta Th1/Th2 e elevou a taxa de sobrevida.(AU)

Assessment of the acid phosphatase CP01850 from Corynebacterium pseudotuberculosis in DNA and subunit vaccine formulations against caseous lymphadenitis / Avaliação da fosfatase ácida recombinante CP01850 de Corynebacterium pseudotuberculosis em formulações vacinais de subunidade e de DNA contra a linfadenite caseosa

The target cp1002_RS01850 from Corynebacterium pseudotuberculosis was used to construct a DNA and recombinant subunit vaccine against caseous lymphadenitis. Recombinant protein rCP01850 was expressed in Escherichia coli using pAE vector, and DNA vaccine was engineered with pTARGET vector. BALB/c mice were divided in five groups containing eight animals each, inoculated with: pTARGET/cp01850 as DNA vaccine (G1); rCP01850 plus Al (OH)3 as recombinant subunit vaccine (G2); pTARGET/cp01850 and a boost with rCP01850 plus Al (OH)3 (G3); pTARGET (G4); or Al (OH)3 (G5). Mice were inoculated and blood samples were collected on days 0, 21, and 42 for the analysis of total IgG, IgG1 and IgG2a by ELISA. In each group, five animals were challenged with Mic-6 C. pseudotuberculosis strain, and three were used for cytokine quantification by qPCR. Although no group has been protected by vaccines against lethal challenge, G2 showed an increase in the survival rate after challenge. Significantly higher levels of IL-4, IL-12, IFN-γ, total IgG, IgG1 and IgG2a were also detected for G2, evidencing a mixed Th1/Th2 immunological profile. In conclusion, despite no protection level provided by different vaccinal strategies using cp1002_RS01850 from C. pseudotuberculosis, G2 developed a Th1/Th2 immune response with an increase in survival rate.(AU)

O alvo cp1002_RS01850 de Corynebacterium pseudotuberculosis foi utilizado para construir uma vacina recombinante de subunidade e de DNA contra a linfadenite caseosa. A proteína recombinante rCP01850 foi expressa em Escherichia coli usando o vetor pAE, e a vacina de DNA foi construída com o vetor pTARGET. Camundongos BALB/c foram divididos em grupos de oito animais, inoculados com: pTARGET/cp01850 como vacina de DNA (G1); rCP01850 e Al (OH)3 como vacina recombinante de subunidade (G2); pTARGET/cp01850 e um boost com rCP01850 e Al (OH)3 (G3); pTARGET (G4); ou Al (OH)3 (G5). Os animais foram inoculados e amostras de sangue foram coletadas nos dias 0, 21, e 42 do experimento para a análise de IgG total, IgG1 e IgG2a por ELISA. De cada grupo, cinco animais foram desafiados com a cepa Mic-6 de C. pseudotuberculosis, e três foram usados para a quantificação de citocinas por qPCR. Apesar de nenhum grupo ter sido protegido pelas vacinas testadas contra o desafio letal, G2 apresentou taxa de sobrevida e níveis de IL-4, IL-12, IFN-γ, IgG total, IgG1 e IgG2a significativamente mais altos, evidenciando um perfil imunológico misto Th1/Th2. Conclui-se que apesar das diferentes estratégias vacinais utilizando cp1002_RS01850 de C. pseudotuberculosis não terem sido capazes de gerar proteção, G2 desenvolveu uma resposta Th1/Th2 e elevou a taxa de sobrevida.(AU)

Condiciones de adsorción de un candidato vacunal basado en vesículas de membrana externa derivada de Salmonella Paratyphi A en adyuvantes de aluminio / Adsorption conditions of a vacunal candidate based on outer membrane vesicles of Salmonella Paratyphi A in aluminum adjuvants

La capacidad inmunoestimuladora de la mayoría de las vacunas es potenciada mediante la adsorción en adyuvantes que contienen aluminio. Variando las condiciones de adsorción (pH, tiempo de adsorción) cambia la cantidad de antígeno adsorbida y por lo tanto la capacidad de estimulación del sistema inmune. El Instituto Finlay de Vacunas investiga un nuevo candidato vacunal basado en vesículas de membrana externa de Salmonella Paratyphi A (VME-SPA). El objetivo de este trabajo fue determinar las condiciones de adsorción de las VME-SPA en dos adyuvantes de sales de aluminio (Al(OH)3 y AlPO4). Para ello, las VME-SPA fueron adsorbidas en ambos adyuvantes bajo diferentes condiciones de pH y tiempo. Mediante la construcción de una Isoterma de Langmuir se determinaron parámetros como la capacidad adsortiva (qm) y el coeficiente de adsorción (Kd). El lote de VME-SPA empleado estaba formado por poblaciones de nanoestructuras con un tamaño de partículas entre 60 y 100 nm. La adsorción de las VME-SPA en ambos adyuvantes, mostró valores ≥95 por ciento a pH neutro (6,5-7,0). Las VME-SPA en presencia de AlPO4 alcanzaron el estado de equilibrio en menor tiempo (99 por ciento a partir de 30 min) en comparación con Al(OH)3 (95 por ciento a partir de 3 h). Las isotermas evaluadas para ambos adyuvantes cumplieron con el modelo de Langmuir (R2≥0,99), con valores de qm y Kd diferentes entre los sistemas de adsorción. El estudio demostró que las VME-SPA se adsorbieron satisfactoriamente en ambos geles, proceso en el que están involucrados diferentes mecanismos de adsorción(AU)

The immunostimulation capacity of most vaccines is enhanced through antigen adsorption on aluminum adjuvants. The changes in adsorption conditions (pH, adsorption time), could change the amount of antigen adsorbed and therefore the ability to stimulate the immune system. The Finlay Institute of Vaccine researches a new vaccine candidate based on outer membrane vesicle from Salmonella Paratyphi A (OMV-SPA). The study aim was to determine adsorption condition of OMV-SPA with two aluminium adjuvants (Al(OH)3 and AlPO4). OMV-SPA was adsorbed in both adjuvants under differences conditions of pH and time. Parameters as adsorptive capacity (qm) and adsorption coefficient (Kd) were determined by construction of Langmuir Isotherm. The lot of OMV-SPA used is composed by population of nanostructure with a particle size between 60 and 100 nm. Adsorption of OMV-SPA in both adjuvants showed values ≥95 percent in neutral pH (6.5-7.0). OMV-SPA with AlPO4 got equilibrium state in less time (99 percent from 30 min) compared with Al(OH)3 (95 percent from 3 h). Isotherms from both adjuvants described Langmuir model (R2≥0.99), with qm and Kd values very different between adsorption systems. As conclusion, the study showed that OMV-SPA was adsorbed satisfactorily in both aluminium adjuvants, process in which are involved different adsorption mechanism(AU)

Evaluation of intranasal and subcutaneous route of immunization in neonatal mice using DODAB-BF as adjuvant with outer membrane vesicles of Neisseria meningitis B.

BACKGROUND: The Neisseria meningitidis bacterium is a Gram-negative diplococcus that can be classified into different serogroups according to the capsular structure. Six of them (A, B, C, W, X, Y) are responsible for causing Invasive Meningococcal Disease (IMD). The strategies for the development of a vaccine for serogroup B have been directed to the use of outer membrane vesicles (OMVs). The aim of this study was to evaluate the immunogenicity of antigenic determinants from OMVs of N. meningitidis B complexed with two different adjuvants: DODAB-BF and aluminum hydroxide (alum), comparing the evaluation of intranasal and subcutaneous route of immunization. METHODS: We used prime-boost immunization for the first time in outbred neonatal mice evaluating the cellular and humoral immune response. RESULTS: Immunoblot, ELISA DOT-ELISA and ELISpot were used universal methods of antibody detection, in order to detect the humoral and cellular immune response in male and female mice. Immunoblot analyzes the specificity of antibodies with the homologous N. meningitidis strain. ELISA served to quantify and compare the titers of antibodies in the serum of mice immunized with DODAB-BF + OMVs and alum + OMVs for IgG, IgG1, and IgG2a. Intranasal immunization produced a mixed response in the T helper cells Th1 and Th2, while subcutaneous immunization exhibited a Th1 profile. The DOT-ELISA identified cross-reactivity with DODAB-BF to different serogroups of N. meningitidis (B, C, W, and Y) that was not observed with alum. ELISpot analyzed IFN-γ- and IL-4 and the results showed the response directly to Th1 and Th2 profile. CONCLUSION: Our findings indicate that DODAB-BF can be an alternative adjuvant for mucosal cell activation with OMVs of N. meningitidis B and that DODAB-BF was similar to aluminum hydroxide as an adjuvant for subcutaneous immunization.

Obtention and characterization of the recombinant simian Interleukin-15 in Escherichia coli for the preclinical assessment of an IL-15-based therapeutic vaccine.

Recombinant simian IL-15 (siIL-15) was obtained for the preclinical assessment of an anti-human IL-15 vaccine. For this purpose, the cDNA from peripheral blood mononuclear cells of a Macaca fascicularis monkey was cloned into a pIL-2 vector. The siIL-15 was expressed in Escherichia coli strain W3110 as an insoluble protein which accounted for 13% of the total cellular proteins. Inclusion bodies were solubilized in an 8 M urea solution, which was purified by ion exchange and reverse phase chromatography up to 92% purity. The protein identity was validated by electrospray ionization-mass spectrometry, confirming the presence of the amino acids which distinguish the siIL-15 from human IL-15. The purified siIL-15 stimulates the proliferation of cytotoxic T-lymphocytes line (CTLL)-2 and Kit 225 cells with EC50 values of 3.1 and 32.5 ng/mL, respectively. Antisera from modified human IL-15-immunized macaques were reactive to human and simian IL-15 in enzyme-linked immunosorbent assays. Moreover, the anti-human IL-15 antibodies from immune sera inhibited siIL-15 activity in CTLL-2 and Kit 225 cells, supporting the activity and purity of recombinant siIL-15. These results indicate that the recombinant siIL-15 is biologically active in two IL-15-dependent cell lines, and it is also suitable for the preclinical evaluation of an IL-15-based therapeutic vaccine.

Comparative efficacy and toxicity of two vaccine candidates against Sporothrix schenckii using either Montanide™ Pet Gel A or aluminum hydroxide adjuvants in mice.

Sporotrichosis is an important zoonosis in Brazil and the most frequent subcutaneous mycosis in Latin America, caused by different Sporothrix species. Currently, there is no effective vaccine available to prevent this disease. In this study, the efficacy and toxicity of the adjuvant Montanide™ Pet Gel A (PGA) formulated with S. schenckii cell wall proteins (ssCWP) was evaluated and compared with that of aluminum hydroxide (AH). Balb/c mice received two subcutaneous doses (1st and 14th days) of either the unadjuvanted or adjuvanted vaccine candidates. On the 21st day, anti-ssCWP antibody levels (ELISA), the phagocytic index, as well as the ex vivo release of IFN-γ, IL-4, and IL-17 by splenocytes and IL-12 by peritoneal macrophages were assessed. Cytotoxicity of the vaccine formulations was evaluated in vitro and by histopathological analysis of the inoculation site. Both adjuvanted vaccine formulations increased anti-ssCWP IgG, IgG1, IgG2a, and IgG3 levels, although IgG2a levels were higher in response to PGA+CWP100, probably contributing to the increase in S. schenckii yeast phagocytosis by macrophages in the opsonophagocytosis assay when using serum from PGA+CWP100-immunized mice. Immunization with AH+CWP100 led to a mixed Th1/Th2/Th17 ex vivo cytokine release profile, while PGA+CWP100 stimulated a preferential Th1/Th2 profile. Moreover, PGA+CWP100 was less cytotoxic in vitro, caused less local toxicity and led to a similar reduction in fungal load in the liver and spleen of S. schenckii- or S. brasiliensis-challenged mice as compared with AH+CWP100. These results suggest that PGA may be an effective and safe adjuvant for a future sporotrichosis vaccine.

Decay of Sabin inactivated poliovirus vaccine (IPV)-boosted poliovirus antibodies.

INTRODUCTION: We conducted a follow-on study to a phase I randomized, controlled trial conducted in Cuba, 2012, to assess the persistence of poliovirus antibodies at 21-22 months following booster dose of Sabin-IPV compared to Salk-IPV in adults who had received multiple doses of oral poliovirus vaccine (OPV) during childhood. METHODS: In 2012, 60 healthy adult males aged 19-23 were randomized to receive one booster dose, of either Sabin-inactivated poliovirus vaccine (Sabin-IPV), adjuvanted Sabin-IPV (aSabin-IPV), or conventional Salk-IPV. In the original study, blood was collected at days 0 (before) and 28 (after vaccination), respectively. In this study, an additional blood sample was collected 21-22 months after vaccination, and tested for neutralizing antibodies to Sabin poliovirus types 1, 2 and 3. RESULTS: We collected sera from 59/60 (98.3%) subjects; 59/59 (100%) remained seropositive to all poliovirus types, 21-22 months after vaccination. The decay curves were very similar among the study groups. Between day 28 and 21-22 months, there was a reduction of ⩾87.4% in median antibody levels for all poliovirus types in all study groups, with no significant differences between the study groups. CONCLUSION: The decay of poliovirus antibodies over a 21-22-month period was similar regardless of the type of booster vaccine used, suggesting the scientific data of Salk IPV long-term persistence and decay may be broadly applicable to Sabin IPV.

Reactogenicity and immunogenicity of inactivated poliovirus vaccine produced from Sabin strains: a phase I Trial in healthy adults in Cuba.

BACKGROUND: To ensure that developing countries have the option to produce inactivated poliovirus vaccine (IPV), the Global Polio Eradication Initiative has promoted the development of an IPV using Sabin poliovirus strains (Sabin IPV). This trial assessed the reactogenicity and immunogenicity of Sabin IPV and adjuvanted Sabin IPV in healthy adults in Cuba. METHODS: This is a randomized, controlled phase I trial, enrolling 60 healthy (previously vaccinated) male human volunteers, aged 19-23 years to receive one dose of either Sabin IPV (20:32:64 DU/dose), adjuvanted Sabin IPV (10:16:32 DU/dose), or conventional Salk IPV (40:8:32 DU/dose). The primary endpoint for reactogenicity relied on monitoring of adverse events. The secondary endpoint measured boosting immune responses (i.e. seroconversion or 4-fold rise) of poliovirus antibody, assessed by neutralization assays. RESULTS: Sixty subjects fulfilled the study requirements. No serious adverse events reported were attributed to trial interventions during the 6-month follow-up period. Twenty-eight days after vaccination, boosting immune responses against poliovirus types 1-3 were between 90% and 100% in all vaccination groups. There was a more than 6-fold increase in median antibody titers between pre- and post-vaccination titers in all vaccination groups. DISCUSSION: Both Sabin IPV and adjuvanted Sabin IPV were well tolerated and immunogenic against all poliovirus serotypes. This result suggests that the aluminum adjuvant may allow a 50% (or higher) dose reduction.

The role of adjuvants in therapeutic protection against paracoccidioidomycosis after immunization with the P10 peptide.

Paracoccidioidomycosis (PCM), a common chronic mycosis in Latin America, is a granulomatous systemic disease caused by the thermo-dimorphic fungus Paracoccidioides brasiliensis. The glycoprotein gp43 is the main antigen target of P. brasiliensis and a 15-mer internal peptide (QTLIAIHTLAIRYAN), known as P10, defines a major CD4(+)-specific T cell epitope. Previous results have indicated that, besides having a preventive role in conventional immunizations prior to challenge with the fungus, protective anti-fungal effects can be induced in P. brasiliensis-infected mice treated with P10 administered with complete Freund's adjuvant (CFA). The peptide elicits an IFN-γ-dependent Th1 immune response and is the main candidate for effective immunotherapy of patients with PCM, as an adjunctive approach to conventional chemotherapy. In the present study we tested the therapeutic effects of P10 combined with different adjuvants [aluminum hydroxide, CFA, flagellin, and the cationic lipid dioctadecyl-dimethylammonium bromide (DODAB)] in BALB/c mice previously infected with the P. brasiliensis Pb18 strain. Significant reductions in the number of colony forming units of the fungus were detected in lungs of mice immunized with P10 associated with the different adjuvants 52 days after infection. Mice treated with DODAB and P10, followed by mice treated with P10 and flagellin, showed the most prominent effects as demonstrated by the lowest numbers of viable yeast cells as well as reductions in granuloma formation and fibrosis. Concomitantly, secretion of IFN-γ and TNF-α, in contrast to interleukin (IL)-4 and IL-10, was enhanced in the lungs of mice immunized with P10 in combination with the tested adjuvants, with the best results observed in mice treated with P10 and DODAB. In conclusion, the present results demonstrate that the co-administration of the synthetic P10 peptide with several adjuvants, particularly DODAB, have significant therapeutic effects in experimental PCM.

Characterizatio of the adsorption process analogous peptides on alumina gel / Carcterización ddelproceso de adsorción de péptidos análogos sobree gel de alúmina / Caracterização do processo de adsorção peptídeos análogos em gel alumnaa

Peptide antigen adsorption on aluminum hydroxide gel must be characterized when formulating vaccines. In this work a peptide belonging to the amino-terminal region of Plasmodium falciparum Merozoite Surface Protein and its analogues have been characterized. The adsorption of 17 analogues on aluminum hydroxide which had greater than 10 mmol/L solubility was quantified at 298 K. Adsorption capacity and affinity constant parameters were calculated by applying the Langmuir's adsorption model. The results have been presented in three groups according to adsorption isotherm trajectory. The first group consists of analogues where the first organization of peptide molecules was presented at low concentrations, followed by a rapid increase in adsorption to high concentrations. The second group consists of analogues having an adsorption pattern showing the formation of a first layer at low peptide concentrations and a second layer at greater concentrations. The third group contains analogues whose adsorption involved the formation of two simple layers, this being differentiated from the second group in that after the second layer had been completed, the amount adsorbed grew notably with increased concentration. The results revealed a more complex pattern that monolayer or bilayer formation. This work constitutes the first approach towards establishing an adsorbed layer structure model using a peptide system.

La adsorción de un antígeno peptídico sobre gel de hidróxido de aluminio debe ser caracterizada para la formulación de vacunas. En este trabajo se caracterizó la adsorción de un péptido que pertenece a la región amino-terminal de la proteína de superficie del merozoite de Plasmodium falciparum y sus análogos. Se cuantificó la adsorción a 298 K sobre hidróxido de aluminio de 17 análogos con una solubilidad mayor de 10 mmol/L. Los parámetros de capacidad de adsorción y constante de afinidad se calcularon aplicando el modelo de adsorción de Langmuir. Los resultados se presentan en tres grupos, de acuerdo con la trayectoria de la isoterma de adsorción. El primer grupo consta de los análogos que presentaron la primera organización de las moléculas de péptido en concentraciones bajas, seguida de un rápido incremento de la adsorción a altas concentraciones. El segundo grupo de análogos tiene un patrón de adsorción que muestra la formación de una primera capa a concentraciones bajas de péptido y una segunda capa a concentraciones mayores. El tercer grupo contiene los análogos cuya adsorción muestra la formación de dos capas simples y se diferencia del segundo grupo en que después de la segunda capa, la cantidad adsorbida crece notablemente con el aumento de la concentración. Los resultados revelaron un patrón de adsorción más complejo que la formación de monocapa o bicapa. Este trabajo constituye la primera aproximación hacia el establecimiento de un modelo de estructura de la capa adsorbida en un sistema peptídico.

A adsorção de um antígeno peptídico sobre um gel de hidróxido de alumíniodeve de ser caracterizado para a formulação de vacinas. Neste estudo foi caracterizada a adsorção de um peptídeo que pertence á região amino-terminal da proteína de superfície do merozoito de Plasmodium falciparum e seus análogos. Foi quantificada a adsorção a 298 K sobre hidróxido de alumínio de 17 análogos com uma solubilidade maior de 10 mmol/L. Os parâmetros de capacidade de adsorção e constante de afinidade foram calculados aplicando o modelo de adsorção de Langmuir. Os resultados sãoapresentados em três grupos de acordo á trajectória da isoterma de adsorção. O primeiro grupo consta dos análogos que apresentaram a primeira organização das moléculas de peptídeoem concentraçõesbaixas, seguido de um rápido incremento da adsorção a altas concentrações. O segundo grupo de análogos tem um padrão de adsorção que mostra a formação de uma primeira camada a concentraçõesbaixas de peptídeo e uma segunda camada a concentraçõesmaiores. O terceiro grupo contém os análogos cuja adsorçãomostra a formação de duas camadas simples e é diferenciado do segundo grupo em que depois da segunda camada, a quantidade adsorvida cresce notavelmente com o aumento da concentração. Os resultados revelaram um padrão de adsorçãomais complexo que a formação de monocamada ou bicamada. Este trabalho constitui a primeira aproximaçãoao estabelecimento de um modelo de estrutura da camada adsorvida num sistema peptídico.

Efficacy of low-level laser therapy and aluminum hydroxide in patients with chemotherapy and radiotherapy-induced oral mucositis

This study evaluated the efficacy of low-level laser therapy (LLLT) and aluminum hydroxide (AH) in the prevention of oral mucositis (OM). A prospective, comparative and non-randomized study was conducted with 25 patients with head and neck cancer subjected to radiotherapy (RT) or radiochemotherapy (RCT). Twelve patients received LLLT (830 nm, 15 mW, 12 J/cm²) daily from the 1st day until the end of RT before each sessions during 5 consecutive days, and the other 13 patients received AH 310 mg/5 mL, 4 times/day, also throughout the duration of RT, including weekends. OM was measured using an oral toxicity scale (OTS) and pain was measured using the visual analogue scale (VAS). EORTC questionnaires were administered to the evaluate impact of OM on quality of life. The LLLT group showed lower mean OTS and VAS scores during the course of RT. A significant difference was observed in pain evaluation in the 13th RT session (p=0.036). In both groups, no interruption of RT was needed. The prophylactic use of both treatments proposed in this study seems to reduce the incidence of severe OM lesions. However, the LLLT was more effective in delaying the appearance of severe OM.

Este estudo avaliou a eficácia da terapia do laser de baixa potência (LBP) e hidróxido de alumínio (HA) na prevenção da mucosite oral (MO). Um estudo prospectivo, comparativo e não-aleatorizado foi conduzido com 25 pacientes com câncer de cabeça e pescoço submetidos a radioterapia (RT) ou radioquimioterapia (RT/QT). Doze pacientes receberam LBP (830 nm, 15 mW, 12 J/cm²) diariamente desde o primeiro dia até o final da RT antes de cada sessão durante 5 dias consecutivos, e os outros 13 pacientes receberam HA 310 mg/5 mL, 4 vezes ao dia, também por toda a duração da RT, incluindo finais de semana. MO foi mensurada usando uma escala de toxicidade oral (ETO) e dor foi mensurada usando a escala visual analógica (EVA). Questionários da EORTC foram administrados para a avaliação do impacto da MO na qualidade de vida. O grupo LBP mostrou menores médias dos escores da ETO e EVA durante o curso da RT. Uma diferença significante foi observada na avaliação da dor na 13ª sessão de RT (p=0,036). Em ambos os grupos, nenhuma interrupção da RT foi necessária. O uso profilático de ambos os tratamentos propostos neste estudo parece reduzir a incidência de lesões severas de MO. No entanto, o LBP foi mais efetivo no atraso do aparecimento da MO severa.

Producción de anticuerpos policlonales IgG contra una proteína con actividad de óxido nítrico sintetasa de Toxoplasma gondii recombinante (NOS-Tg-r) y marcación inmunológica en taquizoítoso / Production of Polyclonal Antibodies against Toxoplasma gondii Recombinant protein with Nitric Oxide Synthase activity and immunologic marking in Tachyzoites

La enzima óxido nítrico sintetasa ha sido estudiada en mamíferos; en los últimos años se ha descrito que existe también en protozoos, pero se desconocen aspectos importantes de su función. Se logró producir anticuerpos policlonales contra la proteína recombinante con actividad de óxido nítrico sintetasa (NOS-Tg-r) de Toxoplasma gondii y realizar marcación inmunológica en taquizoítos. Se usaron dos conejos Nueva Zelanda (Oryctolagus cuniculus)que se inmunizaron por vía intramuscular con NOS-Tg-r, y dos tipos de adyuvantes, hidróxido de aluminio y adyuvante de Freund. Se comprobó la presencia de anticuerpos policlonales con la técnica de ensayo inmunoenzimático indirecto. Los resultados obtenidos mostraron que a NOS-Tg-r con adyuvante de Freund indujo mayor respuesta inmune que la de la NOS-Tg-r con hidróxido aluminio p 0,005). Para verificar si había reacción cruzada, se realizó una prueba ELISA utilizando como antígenos: metaloproteasa de T. gondii recombinante, cisteína proteasa 5 de Entamoeba histolytica recombinante, albúmina al 2%, hidróxido de aluminio y adyuvante de Freund. Los valores obtenidos con sueros preinmunes y contra proteínas alternas no superaron el punto de corte (0,069), lo cual indica que los anticuerpos policlonales obtenidos son específicos para NOS-Tg-r. Se realizó marcación inmunológica en taquizoítos de Toxoplasma gondii con inmunofluorescencia indirecta que mostró una marcación difusa a nivel de citoplasma y confirmó la presencia de esta proteína en los taquizoítos.

The nitric oxide synthase (NOS)is an enzyme well described on mammals but little is known about the role of these enzymes on pathogenic parasites. We produced polyclonal antibodies against a recombinant NOS enzyme from Toxoplasma gondii nd e lso er formed n mmunol locali zation of the enzyme on tachyzoites. We used two New Zealand rabbits (Oryctolagus cuniculus) to perform intramuscular immunization and we used two types of adjuvant: aluminum hydroxide and Freund ’s adjuvant. We tested the generation of polyclonal antibodies by an indirect ELISA assay. The results showed that levels of antibodies were higher in rabbits immunized with Freund ’s adjuvant than with aluminum hydroxide (p =0.005). In order to test cross reactions, we used a recombinant Toxoplasma metallooprotease, a recombinant cysteine protease from Entamoeba histolytica, albumin 2%, hydroxide aluminium and Freund ’s adjuvant as antigens on indirect ELISA assays for the polyclonal serum antibodies. No serum showed absorbances higher than the cutoff level with these antigens, indicating that the polyclonal antibodies were specific for recombinant Toxoplasma NOS. Additionally, we performed immunodetection of the enzyme on Toxoplasma gondii tachyzoites and we obtained a diffuse labeling of the parasite.